Inhibition of isoniazid acetylation



States This invention relates to a therapeutic composition and more particularly to a therapeutic composition including isoniazid and an acetylation inhibitor. This application is a continuation-in-part of copending application Serial No. 625,622, filed December 3, 1956, entitled Inhibition of Isoniazid Acetylation (now issued as United States Patent No. 2,971,887 on February 14, 1961), which is itself a continuation-in-part of applicants copending United States application 452,227, filed August 25, 1954, for an invention of Willard Johnson entitled Therapeutic Composition Including Acetylation Inhibitor (now abandoned).

In application Serial No. 452,227, it was explained that the acetylation of sulfonamides with consequent inactivation and loss of the sulfonamides and possibilities of kidney damage could be inhibited by administering with the sulfonamides a substance which would compete with the sulfonamides for the acetylating enzyme. The inhibition thus caused tends to reduce the danger of kidney damage and, by reducing the rate of urinary excretion makes possible the maintenance of therapeutic blood levels of free sulfonamide, at lower dosage levels and with less frequent administration than was hitherto the case. It Was also disclosed that the acetylation inhibitors inhibited the acetylation of isoniazid.

The inhibition of the acetylation of isoniazid provides a means of attaining greater effectiveness of a given dose of isoniazid by the use of inhibitors of isoniazid acetylation to prevent the inactivation and rapid excretion of the drug. The inhibitors of isoniazid acetylation are able to produce elevated and sustained blood plasma levels of free, therapeutically active isoniazid, thus obviating the necessity of giving increased dosages to obtain higher blood levels of isoniazid. The instant application further develops the use of applicants invention in the acetylation of isoniazid.

isoniazid, as an anti-microbial drug, is considered to be equivalent if not superior to streptomycin in the treatment of pulmonary tuberculosis. However it has been shown by Hughes, J. Pharmacol. and Exper. Therap., 109: 444 (1953), and confirmed by others: Nature, 173: 36 (1954), that when isoniazid is administered to man and monkey, from 50% to 90% of the drug appears in the urine in the acetylated form, that is, as l-is'onicotinyl- Z-acetylhydrazine.

Acetyl-isoniazid has less than one five-hundredth the I activity of free isoniazid against the tubercle bacillus in vitro, while in vivo the parent drug is at least 100 times more active than the acetyl derivative (3). Moreover, as is known to be the case with sulfonamides, the acetylderivative is excreted via the kidney at a much greater rate than is the free drug. Thus, acetylation of isoniazid by the liver leads to rapid inactivation and excretion of the drug, which to some extent accounts for the rapid drop in isoniazid blood levels after the peak blood level has been reached. The over-all eifect of acetylation is in all probability a diminution in the therapeutic eifectiveness of the administered drug.

Both isoniazid and sulfonamides are acetylated by the same enzyme system which is located in the liver of birds to be particularly 3,012,938 Patented Dec. 12, 1961 and mamals (including man). The dual acetylation of isoniazid and sulfanilamide is illustrated by the experiment shown in Table I. isoniazid and sulfanilamide, separately and in combination, were incubated with a cellfree extract of pigeon liver under conditions suitable for TABLE 1 Competition of isoniazid (INH) and sulfanilamide for the same acetylating enzyme in pigeon liver extract Inhibition Inhibition IN H of INH Sulfanilaof sultanll- Additions acetylated, acetylamlde acetylamide m. tion, perated, m. acetylation,

cent percent IN H Sulfanilarnide INl-I, Sulfanilamida- Vessel components-4 ml. of pigeon liver extract; m. potassium phosphate buffer (pH 7.4); 60 um. acetate; 60 m. citrate; 9 am. ATP; 2.92 ,uml INH; 2.61 m. sulfanilamide. Total volume 3 ml.; '90 minutes incubation at 37 C.

The inhibitors used in accordance with this invention as defined in Serial No. 625,622 are soluble, non-toxic compounds selected from the group having the general formula where R is at least one member selected from the group consisting of hydrogen, a halogen, hydroxyl, amino, lower alkyl radicals having not more than three carbon atoms, phenyl, and carboxyl and where A is selected from the group consisting of pyridine, benzamide, benzoic acid hydrazide and benzoylhydroxamic acid and pharmaceutically acceptable salts thereof. The inhibitors used in accordance with the present continuation-impart application are selected from the group consisting of 4arninosal'icylhydroxamic acid, and a-ethyl-thioisonicotinamide and pharmaceutically acceptable salts thereof.

A cell-free extract of pigeon liver was employed for testing the inhibitory effect of various compounds on isoniazid acetylatiom The compounds which were shown effective were p-aminosalicylamid'e, p-aminobenzoic acid and gentisamide, as species for the generic invention defined in Serial No; 625,622, and 5 'bromosalicylhydroxamic' acid, 4'-aminosalicylhydroxamic acid, o-aminoicotinylhydroxamic acid and a-ethyl-thioisonicotinamide as species for the generic invention defined by the present continuation-in-part application. P-aminosalicylic acid is also, as shown by experiments with rabbits, an effective acetylation inhibitor when used in the ratio of A to is isoniazid to paraminosalicylic acid. The results obtained in the in vitro experiments are re produced in Table 2 below.

3 4 TABLE 2 next. As an extra precaution the order of drug administration was reversed; that is, isoniazid alone was given Inhibition of isonilazid (INH) acetylation in vitro 1 48 hours after the dose of lsomazld Plus Experiments were performed to ascertain the relation- 5 ship between the dose level of PAS and the plasma level of free isoniazid. The same three rabbits were used Inhibitor ticorgcfergfiag gelgetent infthroughout this set of experiments. The isoniazid dose g g gl g was kept constant at 50 mg. per k body weight and the hire. sodium PAS dose was varied from 0 to 600 mg. per k". 10 body weight. That is, the isoniazid to sodium PAS -l3romosalieylamide 1. 3X10 52 rati 0 w salicylamide 2 0 5X10 24 o, n a eight basis, varleid fr orn 1.0 to 1.12. Typical 4 36 results are shown in Table it can be seen that there v-cfesotamide igi g g is a progressive increase in the plasma level of free Gentisamide 5X10-4 54 isoniazid at the 1%. hr. and 4 hr. intervals as the dose of M104 74 v pfiydmxybemamidel n 104 22 sod um PAS is increased. Thus, when 1son1az1d and Gamqmidpmidh? 8x104 29 sodium PAS were administered in the ratio of 1:12 the 'f l l 49 plasma level of free isoniazid was 209% of the level atmmosal1eylamlde- 10 3 86 o-Hydroxybenzal isonicotinyl hydrazone 5 70 tamed when ISQIllaZld 310116 Was glven. p-Aminobenzoic Acid 10- 41 fi-mlnoniltlotinlamiglekna 10': 50 pminop eny ace ic oi 2X10- 36 4-Amino-5-imidazole-carboxamlde.-. 2X10" 31 TABLE 4 Pyrazinamide 8X10" 19 ls-lilydligzigophthala mg 2 10 53 d h aioy y roxamic ci 5X10- 32 0 5-Bromosalieylhydroxamic Acid 2. 5X10- 56 Efiect of Increasing l t e 2 plasma levels i-Aminosalicylhydroxamie Acid 2.5 10- as f 1 ee Isonlazld rabblts fi-Aminonicotinylhydroxamic Acid. 10" 58 a-Ethyl-th ioisonicotinamlde 10- 50 Dosage mgjlrgJ Bloodplasmalevels Percent 1 Pigeon liver acetylating system (see W. Johnson, Canad. J. Bioehem. Ptgysiol 35:39? t1935). The INH concentration was 8X10 M unless body INH/ ag -33133? ggy??? o erwisem me e Expt. Sodium isomazid 3 In these experiments the INH concentration was 7.75X10" M. 3 PAS plasma SOdlLlIll ratio at lnterafter level at INH PAS dosage of ihour vals, I a u I 1 Table 3 shows 2 experiments in which SOdllllIl PAS 14 mterval was employed as an inhibitor of isoniazid acetylation.

0 0 1:0 1.7 0. 45 In the first experiment 2 rabbits, each weighing 6 kg., 2 50 m 67 49 c 50 250 1:5 2. 45 0.86 91 were given 300 m of isoniazid by stomach tube. Forty 50 600 1:12 63 1 39 209 eight hours later the same rabbits were given 300 mg. of Isomazld pl-us of sodmm PAS l plasma "The same 3 rabbits, each weighing 3 kg., were used for every levels of free isomazid were determined at intervals of experiment 11%;, 21A d 4 h r ft d I b th bbit th m Free isongazidjbbood plasma levels as shown are average values for administration of PAS with isomazid resulted in an en- Pgmupo m is hanced plasma level of free isoniazid. This effect of PAS was most striking at the four-hour interval, when the plasma level of free isoniazid was more than twofold The effect of 5-bromosalicylhydroxamic acid on plasma that of the control level. A similar eifect of PAS is levels of isoniazid was tested in 7 rabbits, each rabbit shown in experiment 2 of Table 3. acting as its own control. The results, shown below in TABLE 3 Efiect of p-aminosalicylic acid (sodium) on blood plasma levels of free isoniazid (INH) in rabbits following a single oral dose Blood Plasma Levels of Free Isoniazld Expt. Rabbit Weight, Drug Dose (mgJkg. body wt.) 1% hrs. 7 2% hrs. 4 hrs.

No. No. kg.

mg. Pereentof mg. Percentof mg. Percent-oi percent control percent control percent control 1 6 Control (INH) 4. 8 1. 4

1 INH50+PAS (So um) 500.. 6.3 2.9

"""" 2 6 Control (INH50) 3.1 1.1

"""" 4 3 Control (I 50) 2. 24 0.64 INH50+PAS (Sodium) 600"." 2. 86 1. 86

It should be mentioned that 24 hours after the ad- Table 5, indicate that the administration of INH in comministration of a single dose of isoniazid there is no bination with S-BSH gives rise to an appreciable increase detectable sign of the drug in the bloodstream, and hence in plasma levels, of free INH, over that which is ohno carry-over of isoniazid from one experiment to the tained when INH alone is administered.

TABLE 6 g INH 1 alone and 4 hour g. percent in c mbinati n with 5-BSH 2 Percent m of control INH blood levels in patients receivin (S-BSH) on 2 hour g. per-, cent Blood Plasma Levels of Free IN H 1 s to lsonlazld, PAS to para-amino salicyclic acid, PABA d e 2628462826686 m .m e 31%0213031101 m m r M g m IPa v m a 1 b M S a 688666462264622462646 m m m m ruoLtLaoaanooaaaoLLdt L S m 1 452 2 LZtLLZZztitzLLttzattttztzzLz m m e Hm il V e N a d m W 64842482452 m I t e c w 10 001 000 201 m n s r r S A v. e u m m P m H mm 0 M 050050055005000055505050050050-M0 m d w a 7 m cm 848282618442284624424 LO LLQLZQQLLQZLLLQQQZQLQLZQLLQLQL M w I f. l mm m a m m m mam i t I S R Dr. W mm m M A m m u m T t d hf n m P r 4 QM 363636363666636366666 .m e f t a m.m am mn P w w m M mme m 0 mdupw i0 .1 B a mm v. mm e P mme d .IOM :fi Tm m 0 mm .& n b L BLUT 6 V n3 12 t n 08 n n u ei m h m h h n bd 0 0 0 3 4 5 a u m n m m m r +n m u m u m. g o m 9 a 9 no u s m f en m m ne m m m. m m 5 n mwm j 2 P ww Q Q Q Q W 88 1 2 3 4 5 1 Percent m of control TABLE 5 cylhydroxamic acid of free ison'iazid '(INH) in rabb lhour mg. percent Effect of 5 -bromosz zli blood plasma levels following a single oral dose Drug Dose (mg/kg. body Wt.)

(INH 50).- =BSH 500 STUDIES IN HUMANS 1 Average of 7 rabbits.

The eflect of S-bromosalicylhydroxarrdc acid (S-BSH) on plasma levels of isoniazid (INH) as observed in rabbits No. of animals 7 Control INH50+5 INH alone-Patients received their usual therapeutic doses of INH (300-400 mg. per day) for 3 days th INH+5-BSH.Patients received 300-400 mg. of INH 35 and 3 grams S-BSH for 3 days prior to the test. On the It should be noted that each patient served as his own control. The results, shown in Fable 6, indicate that has been corroborated in 32 human subjects (at the Royal 25 Edward Laurentian Hospital, Montreal, Quebec, Canada). The experimental conditions were as follows:

e test. On the day of the test they received 150 INH at 8:00 am. and 3 hours later blood was taken INH determination.

day of the test they received 150 mg. INH plus 1 gram SBSH at 8:00 am. and blood was taken 3 hours later for INH determination.

S-bromosalicylhydroxamic acid produced a fourfold increase in isoniazid blood concentration in 9 patients and 45 a two fold increase in 23 patients. No patient failed to show an increase in the blood concentration of isoniazid.

In the above table INH refer to para-aminobenzolc acid and PAS-amide to p-amlnosallcylamide.

While the ratio of isoniazid to acetylation inhibitor may vary widely, satisfactory results are normally achieved through the use of ratios of isoniazid to acetylation inhibitor of 1:5 to 1:30.

Thus, when the inhibitor is S-bromosalicylhydroxamic acid a suitable dose for an adult would beat tablet containing as its active ingredients 50 mg. of isoniazid and 500 mg. of 5-bromosa1icylhydroxamic acid to be administered several times daily.

I claim:

1. A therapeutic composition comprising as its active ingredients isoniazid and an acety-lation inhibitor selected from the group consisting of:

(a) 4-aminosa1icy1hydroxamic acid, and

(b) a-ethyl-thioisonicotinamide,and the pharmaceuti- '15 cally acceptable salts thereof.

2. A therapeutic composition comprising as its active ingredients isoniazid and 4-aminosalicylhydroxamic acid in a ratio of between 1:5 and 1:30.

3. A therapeutic composition comprising as its active ingredients isoni azid and a-ethyl-thioisonicotinamide in a ratio of between 1:3 and 1:15.

4. A therapeutic composition comprising as its active ingredients isoniazid and the pharmaceutically acceptable salts of a-ethyl-thioisonicotinamide in a ratio of between 10 1:3 and'1z15.

References Cited in the file of this patent UNITED STATES PATENTS 2,937,116 Johnson May 17, 1960 

1. A THERAPEUTIC COMPOSITION COMPRISING AS ITS ACTIVE INGREDIENTS ISONIAZID AND AN ACETYLATION INHIBITOR SELECTED FROM THE GROUP CONSISTING OF: (A) 4-AMINOSALICYLHDROXAMIC ACID, AND (B) A-ETHL-THIOISONICOTINAMIDE, AND THE PHARACEUTICALLY ACCEPTABLE SALTS THEREOF. 